Radikal Therapeutics (RTX) is developing a novel bifunctional small molecule, R-503, intended for the prevention of lung ischemia-reperfusion injury (LIRI) associated with orthotopic lung transplantation. LIRI results from an acute reperfusion-induced alteration in the synthesis of the free radical superoxide anion that is subsequently converted into powerful toxins, including peroxynitrite, hydrogen peroxide, and hydroxyl radical. These oxidizing and nitrosating species induce DNA single strand breaks that activate poly(ADP-ribose) polymerase (PARP), a nuclear DNA repair enzyme that in turn depletes its substrate NAD, resulting in an exhaustion of intracellular energetics, ATP depletion, and tissue necrosis. PARP activation also induces a widespread expresion of pro-inflammatory genes that contribute to vascular permeability, lung edema, neutrophil infiltration, pulmonary shunt, and respiratory failure. In experimental models of LIRI injury, pharmacologic inhibition of PARP potently reduces lung injury, but the extent of protection was not complete and its successful clinical translation is uncertain. We now propose a more thorough interruption of LIRI-induced tissue damage, exploiting the simultaneous and synergistic removal of upstream DNA-damaging oxidizing and nitrosating species plus downstream PARP inhibition. R-503 is formed from the covalent linkage of 2 moieties, each with demonstrated tissue protection: 1) a PARP inhibitor moiety, and 2) a thiol-rich dihydrolipoyl (DHL) domain that acts as a broad-spectrum redox catalyst. The inclusion of the DHL moiety confers unique properties on R-503, allowing the molecule to shut down both PARP-dependent and PARP-independent pathways of redox stress. R-503 is a potent PARP inhibitor (IC50=20 nM), more cytoprotective than a monofunctional PARP inhibitor in vitro, and remarkably protective in vivo, as shown in murine LD100 models of zymosan-induced multiple organ failure and chlorine inhalational lung injury. Specific Aim: Establish the superiority and in vivo synergy of the bifunctional PARP inhibitor R-503 in an experimental rat model of LIRI. Prior to 90 min of unilateral lung ischemia and 4 h of reperfusion, rats will be treated with IV R-503, DHL, a monofunctional PARP inhibitor (INO-1001), or a combination of DHL and INO-1001. A sham animal will not undergo ischemia nor receive drug therapy. We expect that R-503 will exhibit superior efficacy, relative to treatment with INO-1001, DHL, and their combination, with respect to: 1) tissue damage (histology score, levels of neutrophil infiltration, lipid peroxidation, protein nitrosation, vascular permeability, PARP activation, and apoptosis) and arterial oxygenation; and 2) bronchoalveolar lavage markers of pulmonary injury (levels of neutrophil infiltration, concentrations of TNF-, MIP-1, and nuclear NF-?B). Such treatment effects are expected to translate into clinical endpoints in LIRI of decreased: 1) pulmonary shunt, 2) duration of mechanical ventilation, 3) length of hospitalization, and 4) all-cause 30 and 365 day mortality. PUBLIC HEALTH RELEVANCE: Ischemia-reperfusion injury is a major medical complication following lung transplantation and contributes to the high mortality in this population. At present there are no approved prophylactic measures. We are developing a novel drug that targets the basic mechanisms of this condition and will test this agent in a clinically-relevant animal model.